The present invention relates generally to detection of and vaccination against Feline immunodeficiency virus (FIV). More particularly, the invention relates to a highly cytopathic and infectious proviral clone constructed from the genomic DNA of a Pallas""s cat FIV. The nucleotide sequences, antigens and chimeric viruses derived from the reconstructed clone can be used for the detection of and protection against FIV.
Feline immunodeficiency virus, (FIV) a lentivirus of cats is associated with feline acquired immunodeficiency syndrome (AIDS) (see Pederson et al., 1987, Science 235:790). Under natural conditions, cats experience an asymptomatic carrier state for years following initial FIV infection before developing an AIDS like disease. Cats experimentally infected with FIV exhibit signs of acute infection which resolve over a few months. Disorders associated with FIV infection include abortion, alopecia, anemia, gingivitis/stomatitis, upper respiratory infections, chronic enteritis, diarrhea, neurological abnormalities, and recurrent ocular disease, see R. English et al., 1990, J. Am. Vet. Med. Assoc., 196:116; N. Pederson et al., 1989, Vet. Immonol. Immunopathol. 21:111, J. Yamamoto et al., 1989, J. Am Vet. Med. Assoc. 194:213.
FIV and the human immunodeficiency virus, HIV-1, belong to the lentivirus subfamily of retroviruses and have similar morphology, protein composition and Mg++ dependency of their reverse transcriptases (RT). Pederson et al., 1987, supra; Pederson et al., 1989, supra. They both display tropism for T lymphocytes and monocytes and are capable inducing these cells to form syncytia (see Brunner and Pederson, 1989, J. Virol. 63:5483). The etiology and pathogenesis of FIV infection closely resembles those of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), which cause acquired immunodeficiency syndrome in humans and primates respectively. Thus, FIV infection in cats provides a valuable animal model for human immunodeficiency virus-1 (HIV-1) induced AIDS. The pathogenesis of HIV-1 infection has been attributed to virus-induced reduction of CD4+ lymphocyte numbers and function, resulting in decreased immune responsiveness and subsequently severe secondary infections (see M. McChesney and M. Oldstone, 1989, Ad. Immunol., 4:335).
The discovery of feline T-lymphotrophic lentivirus (now known as Feline Immunodeficiency virus) was first reported by Pederson et al., 1987, supra at 790-793. Cloning and sequence analysis of FIV have been reported by Olmsted et al., 1989. Proc Natl. Acad. Sci. USA. 86:4355-4360; and Talbott et al., 1989, Proc. Natl. Acad. Sci., USA 86:5743-5747. Molecular clones of several domestic cat isolates of FIV have been sequenced (Maki et al., 1992, Arch. Virol. 123:29-45; Miyazawa et al., 1991 J. Gen Virol. 74:1573-1580; Olmsted et al., 1989, Proc. Natl. Acad. Sci. USA, 86:2448-2452; Phillips et al., 1990 J. Virol. 64:4605-4613; and Talbott et al., 1989, Proc. Natl. Acad. Sci. USA, 86:5743-5747). However, the full nucleotide sequence of only two non-domestic cat lentivirus (isolated from a puma and a Pallas"" cat) has been reported (Langley et al., 1994, Virology 202:853-864; Barr et al., 1995 J. Virol. 69:7371-7374; deposited as accession no. U56928). Nucleotide sequence data from short regions of the pol gene have been obtained for lentivirus infecting additional pumas, and lion (Brown et al., 1994, J. Virol. 68:5953-5968; Olmsted et al., 1992, J. Virol. 66:6008-6018). The FIV provirus includes the structural genes for group-specific antigens (gag gene), envelope proteins (env gene) and reverse transcriptase (pol gene), as well as several short open reading frames similar to those of other lentiviruses. The gag gene of FIV has been reported to encode a polypeptide of about 450 amino acids, which undergoes posttranslational modification. (Talbott et al, 1989, supra; Phillips et al., 1990, supra). The gag gene is thought to be highly conserved among FIV strains (Phillips et al., 1990, supra).
Based on the available cloning and sequencing analysis data, the various species of cats appear to be infected with their own unique lentiviruses. This is similar to the significant strain differences noted among human (Oram et al. 1990xe2x80x94AIDS Res. Hum. Retroviruses 6:1073-1078) and simian (Fomsgaard et al 1991, Virology 182:397-402) immunodeficiency virus isolates. These differences have an impact on the diagnostic procedures, therapeutics and vaccines, making the task of developing broad-spectrum vaccines or detection systems more difficult. Similarly, because FIV isolates from domestic cats exhibit heterogeneity at both the cellular and molecular level (Miyazawa et al., 1991, supra; Phillips et al., 1991, supra), and because these differences are more pronounced between isolates of FIV from domestic and nondomestic cats, vaccines and detection systems for screening various species of domestic and nondomestic cats are not currently available.
In the past, FIV antigens have been used to elicit antibodies which may protect a cat against virus infection and/or replication. These antigens include the FIV gag protein and the env protein. However, these antigens are typically not cross reactive with antibodies from other species and hence are not expected to protect a broad range of species. It would be desirable to identify antigens that have a broad specificity, and as a result cross react with antibodies from different species of cat. Such antigens would be useful for detection and/or immunization purposes.
It would also be useful to identify FIV related viruses that can be used as antigens for a broad range of species of cats. None of the isolated FIVs express broad specificity polypeptides. Thus, it would be useful to construct chimeric viruses expressing polypeptides of desired specificity. Shibata et al. 1991, J. Virol. 65:3514-3522, reported the preparation of a chimeric virus containing HIV-1 tat, rev, and env genes in a SIV provirus. The SIV provirus did not contain functional vpr and nef genes, which are considered to be non essential for viral replication and infection of tissue cultured cells. The chimeric viruses replicated in macaque peripheral blood mononuclear cells. However, when used for infecting macaques, the level of virus replication was low and the infection did not persist beyond two months (see U.S. Pat. No. 5,664,195). Thus, the construction of chimeric viruses having desired biological properties like high immunogenicity and low cytopathicity has as yet not met with much success.
A Pallas""s cat FIV isolate (FIV-Oma) was observed to elicit a unique immune response in domestic cats. After an initial seropositive period, the cats had undetectable levels of antibodies in their serum. A highly cytopathic and infectious clone (FIV-Oma3) has been constructed from the genomic library of this FIV. The recombinant virus of the present invention is highly cytopathic and infectious in culture.
Antigens from the Pallas""s cat FIV (FIV-Oma) and from the recombinant virus, FIV-Oma3, have been observed to have a broad specificity for various species of FIVs, in contrast to most of the antigens from other domestic or non-domestic cats. Hence, it is an object of the present invention to provide antigens having a broad specificity for immunization of cats.
Another object of the present invention is to provide a detection system based on the antigens having a broad specificity that will identify FIV infection in both domestic and non-domestic cats.
A further object of the present invention is to provide one or more nucleic acid sequences, encoding for FIV polypeptide(s) which can be used as probes for the detection of FIVs and can be inserted for expression into recombinant viral vectors.
A still further object of the present invention is to provide a system for evaluation of therapeutic agents that inhibit the cytopathic effects of lentiviruses.
The recombinant virus, FIV-Oma3 is highly cytopathic in culture. This clone can be used to identify gene sequences that are involved in conferring immunogenicity and cytopathicity in FIV strains. Further, chimeric viruses can be constructed which are immunogenic and highly cytopathic in culture. Alternatively, chimeric viruses can be constructed that are pathogenic in cats. Thus, one further object of the present invention is to provide chimeric viruses having the desired combination of genes which can be used as vaccines to induce antibodies to protect against virus infection and/or replication.